WO2004096195A2

WO2004096195A2 - Use of ppar-gamma agonists as anti-infective agents

Info

Publication number: WO2004096195A2
Application number: PCT/FR2004/001018
Authority: WO
Inventors: Agnès COSTE; Bernard Pipy
Original assignee: Universite Paul Sabatier Toulouse Iii
Priority date: 2003-04-28
Filing date: 2004-04-27
Publication date: 2004-11-11
Also published as: EP1628646A2; EP1628646B1; WO2004096195A8; WO2004096195A3; FR2854078B1; DE602004028116D1; FR2854078A1

Abstract

The invention relates to a method of producing a therapeutic composition that stimulates the innate immune response by induction and/or amplification of the expression of macrophage mannose receptors. Advantageously, the invention is applicable for preventive or curative treatments for infectious diseases of bacterial, fungal, protozoan, etc. origin and is of particular interest in the treatment of immunodepressed patients. According to the invention, in order to induce and/or amplify the expression of the macrophage mannose receptor, the aforementioned macrophages are brought into contact with an effective quantity of at least one agonist ligand of the PPAR< nuclear receptor.

Description

INDUCTION OF MANNOSE RECEPTOR EXPRESSION OF MACROPHAGES, AND APPLICATIONS

The invention relates to a method for manufacturing a therapeutic composition, stimulating the innate immune response by induction and / or amplification of the expression of mannose receptors of macrophages. The invention is advantageously part of the preventive or curative treatment of infectious diseases of bacterial, fungal, protozoan origin ... and is of particular interest in the medication of immunocompromised subjects. The innate immune response, essential in the body's defense mechanisms against infectious agents, is an early immune reaction that starts at the first moments of infection. Macrophages participate in this innate immune response by recognizing, phagocytosing and destroying the infectious agent. The recognition phase consists of the identification of superficial biochemical structures characteristic of pathogens and involves a number of receptors on the surface of macrophages.

• Among these receptors, the mannose receptor (we also speak of a mannose receptor) allows the recognition, but also the fixation and phagocytosis, of a wide variety of pathogens, in particular pathogens of bacterial, fungal and protozoan origin. , which have on the outer face of their walls glycoproteins, polysaccharides and / or lipopolysaccharides having at their free ends mannosylated and / or fucosylated units. For simplification purposes, in the rest of the text, these pathogens will be designated by "pathogens with mannosylated and / or fucosylated motifs". The involvement of the macrophage mannose receptor (MMR) is thus essential in the body's defense processes. However, it is only effective if this receptor is strongly expressed on the surface of macrophages.

However, although the role of the mannose receptor of macrophages is today well defined, the ways of regulating the expression of this receptor are still poorly understood. It is the same for the biochemical actors involved in the induction of the expression of this receptor. In this context, the main objective of the invention is to provide compounds capable of inducing and / or amplifying the expression of MMR.

It thus aims to highlight compounds capable of improving alertness and / or the response of the immune system, with regard to numerous microorganisms via recognition by the mannose receptors of macrophages.

Throughout the text, the term "biological medium" means a medium of biological composition, that is to say comprising living matter, in particular monocellular and / or multicellular organisms. A biological medium can be artificial (for example, a culture medium), or natural (for example, a living tissue, a blood composition, isolated or not from a multicellular organism -in particular a-primate-).

Furthermore, the term “macrophage” denotes any cell, essentially of tissue localization, resulting from the differentiation of monocytes (macrophages as such, dendritic cells, phagocytic neutrophil cells) as well as monocytes. "Macrophage" in the sense of the invention thus generally designates any cell capable of expressing MMR and having the capacity to phagocyte.

The invention also aims to propose new perspectives in the therapeutic treatment of infectious diseases - in particular opportunistic diseases -, in particular in the context of the medication of immunocompromised subjects.

The invention therefore relates, firstly, to a method of stimulating the expression of mannose receptors of macrophages in which, an effective amount of at least one agonist ligand of PPARγ is treated, a biological, extracorporeal or intracorporeal medium , including macrophages. The term "effective amount" means an amount capable of inducing and / or amplifying the expression of the mannose receptors of at least part of the macrophages of the target biological medium.

The invention is thus based on the surprising and hitherto unsuspected observation that the activation of the nuclear receptor PPARγ leads to gene induction and protein expression of MMR. According to Vamecq J. and Latruffe N., 1999 (The Lancet, 354: 141-148), a document summarizing the general knowledge concerning PPARs (receptors for peroxisome proliferators), PPARγ is implicated in adipogenesis phenomena, cell differentiation and regulation of certain anti-inflammatory reactions. Although mainly expressed in fat cells, PPARγ has also been detected in macrophages.

MMRs are not discussed in this publication.

So far, there has been no reason to suspect any involvement of PPARγ in the mechanisms of stimulation and / or amplification of MMR expression.

The highlighting by the inventors of the functional link existing between the transcription factor PPARγ and the expression of MMR, thus opens up new perspectives for application to agonist ligands of PPARγ.

Advantageously, at least one of the agonist ligands of PPARγ used to induce and / or amplify the expression of MMR according to the invention is chosen from the group of natural ligands: 15d-PGJ ₂ , prostaglandins A1 and D2, acids 9- and 13-hydoxyoctadecanoic, LDL derivatives

(low density lipoproteins) oxidized.

Among these natural ligands, 15d-PGJ ₂ (15-deoxy-Δ ¹² ^'14 - prostaglandin J ₂ ), a metabolite of prostaglandin D2 (PGD2), is known to be one of the most powerful agonist ligands of PPARγ.

It is also and advantageously possible to choose at least one of the agonist ligands of PPARγ from the group of synthetic ligands: troglitazone (Resulin®), rosiglitazone (Avandia®), pioglitazone (Actos®), ciglitazone, englitazone, GW 347845, KRP-297, JTT-501B, SB 213068, GI 262570, GW 1929, GW 7845, GW 0207, L-796449, indomethacin, ibuprofen.

Troglitazone, rosiglitazone, pioglitazone, ciglitazone, englitazone, GW 347845 belong to the family of thiazolidinediones and are commonly used in the medical field as lipid-lowering drugs for the treatment of certain diabetes. Likewise, indomethacin and ibuprofen are commonly used as non-steroidal anti-inflammatory drugs for the treatment of certain inflammatory diseases, such as atherosclerosis and osteoarthritis.

In particular, WO 03/059271 teaches the use of agonist compounds of PPARγ to treat or prevent inflammations, cancers, cardiovascular diseases ... WO 03/059271 does not deal with MMR and as regards methods and the therapeutic compositions described, they are only envisaged as remedies for the symptoms of certain inflammations. There is neither described nor suggested any biological effect of the agonist ligands of PPARγ on the causes of these inflammations, in particular on possible infectious agents capable of generating some of these inflammatory reactions.

Thus, never before has it been envisaged to use these same compounds to stimulate and / or amplify the expression of the mannose receptors of the macrophages and even less to obtain the triggering of the effector functions of the macrophages (phagocytosis, synthesis of cytokines and oxidizing agents, presentation of the antigen) against pathogens of bacterial, fungal or protozoan origin.

By using Candida albicans (C. albicans) as a model of a microorganism sensitive to MMR, the inventors were able to determine that, beyond a simple modulation of the expression of MMR, the treatment of macrophages with an agonist ligand of PPARγ leads to a remarkable stimulation of the ability of macrophages to recognize and eliminate the pathogen, not only by phagocytosis, but also by an increased production of oxidizing agents. The inventors have thus managed to demonstrate that the induction of the mannose receptor by the agonist ligands of PPARγ allows the elimination of a fungal infection of the gastrointestinal tract by C. albicans. This elimination takes place in the absence of antibodies or an adaptive immune response. The anti-infectious effect of agonist ligands of PPARγ is the consequence, on the one hand, of the increase in phagocytosis of C. albicans mediated by the mannose receptor and, on the other hand, of the death of C. albicans caused by reactive oxygen intermediates (IROs). These are secreted by macrophages at during yeast phagocytosis. In fact, it has been shown that the candidicidal function of macrophages disappears when the superoxide anion is suppressed by superoxide dismutase (SOD) and that the production of reactive oxygen species by macrophages is suppressed by the antisense of the messenger RNA coding for MMR (Coste A. et al, Immunity, 2003, 19: 329-339). These lessons which flow from the work of the inventors, as well as the invention, are completely independent of the anti-inflammatory properties of PPARγ.

This capacity which the agonist ligands of PPARγ have to stimulate the expression of the mannose receptors and therefore, on the one hand, to reinforce the vigilance of the macrophages with regard to infectious microorganisms with mannosylated and / or fucosylated motifs and, on the other hand, to trigger the ^~ effector functions of macrophages, when these are in the presence of this type of pathogen, can be advantageously used for the medication of subjects with immune deficiencies, innate or acquired. For certain immunocompromised subjects, such as people with AIDS or patients treated with chemotherapy (for example, patients with cancer or hemopathy) or with immunosuppressants (for example, after a transplant), induction and / or the amplification of the expression MMR would make it possible to effectively compensate for the insufficiency of their immune system, by improving not only the vigilance of the innate immune system with regard to numerous infectious microorganisms, but also by stimulating and amplifying the functions effectors of macrophages in order to obtain an effective immune response, of the Th2 type. Optionally, the induction and / or amplification of the expression of MMRs will make it possible to redirect the immune defense towards a Th2 type response to compensate for a possibly defective Th1 type response.

In this context of stimulation of the immune system via the expression of MMRs, the invention is addressed in particular to immunocompromised subjects. It is also intended for people who may be exposed to infectious agents with mannosylated and / or fucosylated patterns on the outside of their walls, or who have been infected with them. Examples of infectious agents targeted by the invention include: Streptococcus pneumoniae, Klebsellia pneumoniae, Cryptococcus neoformans, Mycobacterium tuberculosis, Candida albicans, Leishmania donovani, Pneumocystis carinii, Trypanosoma cruzi, Aspergillus fumigatus, pseudomonas aeruginosas, Legionella pneumophilla. The stimulation of the immune system of an individual - in particular, an immunocompromised individual -, by amplification of the expression of MMR, can be carried out by administering to the latter an effective amount of at least one agonist ligand of PPARγ , capable of inducing a distribution of ligand (s) in the target biological medium, at an effective concentration, that is to say capable of inducing and / or amplifying the expression of MMRs of at least part of the macrophages present in this medium - for example, a tissue or a composition of peripheral blood -. This concentration is, in general, at least equivalent to 1ΕC50 of the ligand (s) used (s). The biological medium containing macrophages according to the invention corresponds, in this particular case, to a natural intracorporeal medium. Table 1 below presents the EC50 values for certain synthetic agonist ligands of PPARγ.

Table 1: Transactivating activity of PPARγ agonists (CE IN - N ° 70, March / April 2002)

Depending on the target site, administration can be by oral, rectal, or parenteral (subcutaneous, transdermal, intravenous, intramuscular) route. Furthermore, depending on the target site, the distribution of the active compound can flow to the target via the peripheral blood system, or reach it by topical administration (administration of the compound directly in contact with the cell site to be treated). The dosage form and the doses to be administered will be chosen accordingly.

Taking into account the EC50 value of some of the agonist ligands of PPARγ currently known, it is possible to envisage, in particular for the compounds cited above, use at effective doses without risk of toxicity (for example, rosiglitazone may be prescribed at doses daily of the order of 4 to 8 mg for an adult weighing 70 kg). It should be noted that most of the agonist ligands of PPARγ, in particular those previously cited, have already successfully passed the toxicity tests within the framework of obtaining the marketing authorization, as anti-inflammatory or lipid-lowering agents.

According to an alternative embodiment, in accordance with the invention and also coming within the framework of the stimulation of the immune system by induction of the expression of MMR in an individual - in particular immunosuppressed -, the biological medium comprising macrophages to be stimulated can be also extracorporeal. The macrophages are then prepared from a blood sample from a patient, then are treated with an effective amount of at least one agonist ligand of PPARγ, before being reinjected into this same patient.

Optionally, the macrophages can also come from a donor immunocompatible with the recipient patient. In particular, the latter is affected by immune deficiencies, in particular a deficit in mature macrophages.

According to this same perspective of stimulation of the expression of MMR, the invention extends to a method of treatment of a living tissue or a composition of peripheral blood, not isolated, of a living primate -in particular man-, in which is administered into a tissue or a peripheral blood composition of said primate, an effective amount of at least one agonist ligand of PPARγ, adapted to induce a distribution of ligand (s) in said tissue or said composition peripheral blood, at a therapeutically effective concentration, that is to say capable of inducing and / or amplifying the expression of the MMRs of the macrophages present in said tissue or said peripheral blood composition.

According to another aspect, the invention relates to a process for the elimination, intracorporeal or extracorporeal, of microorganisms of bacterial, fungal or protozoan origin, having, on the external face of their walls, polysaccharides and / or lipopolysaccharides with mannosylated and / or fucosylated free ends. According to the invention, which is placed, in a medium infected with said microorganisms and in contact with macrophages, an effective amount of at least one agonist ligand of PPARγ as an induction and / or amplification factor of the expression of mannose receptors of macrophages.

Advantageously and according to the invention, said microorganisms are chosen from: Streptococcus pneumoniae, Klebsellia pneumoniae, Cryptococcus neoformans, Mycobacterium tuberculosis, Candida albicans, Leishmania donovani, Pneumocystis carinii, Trypanosoma cruzi, Aspergillus fumigatus, pseudomon.

Advantageously and according to the invention, at least one of the agonist ligands of PPARγ is chosen from the group of natural ligands: 15d-PGJ ₂ , prostaglandins Al and D2, 9- and 13-hydoxyoctadecanoic acids, derivatives of oxidized LDL . This ligand can also be chosen from the group of synthetic ligands: troglitazone, rosiglitazone, pioglitazone, ciglitazone, englitazone, GW 347845, KRP-297, JTT-501β, SB 213068, GI 262570 , GW 1929, GW 7845, GW 0207, L-796449, indomethacin and ibuprofen. According to the invention, the use of a PPARγ agonist ligand to eliminate an infectious microorganism via stimulated macrophages, may interest an infected artificial medium, for example a culture medium, or an infected natural medium, for example a living tissue or peripheral blood, isolated or not from a multicellular organism -in particular a primate, in particular living-. The invention therefore also extends to a method of manufacturing a therapeutic composition comprising at least one ligand PPARγ agonist as active compound capable of inducing and / or amplifying the expression of mannose receptors of macrophages.

More particularly, it is a process for manufacturing a therapeutic composition - in particular an immunostimulatory composition - intended for the preventive or curative treatment of a pathology belonging to the group of pathologies induced by microorganisms, of bacterial origin. , fungal or protozoan which have mannosylated and / or fucosylated motifs on the outside of their walls, with the exception of Mycobacterium avium.

According to the invention, a therapeutically effective amount of at least one agonist ligand of the nuclear receptor PPARγ is used as an induction and / or amplification factor for the expression of MMRs.

Advantageously and according to the invention, these are microorganisms chosen from: Streptococcus pneumoniae, Klebsellia pneumoniae, Ciyptococcus neoformans, Mycobacterium tuberculosis, Candida albicans, Leishmania donovani, Pneumocystis carinii, Trypanosoma cruzi, Aspergillus fumigatus, Pseudomonon.

Advantageously and according to the invention, there is used for the manufacture of said therapeutic composition at least one agonist ligand of

PPARγ chosen from the group of natural ligands: 15d-PGJ ₂ , prostaglandins Al and D2, 9- and 13-hydoxyoctadecanoic acids, derivatives of oxidized LDL.

For the manufacture of a therapeutic composition in accordance with the invention, this ligand can also be chosen from the group of synthetic ligands: troglitazone, rosiglitazone, pioglitazone, ciglitazone, englitazone, GW 347845, KRP- 297, JTT-501β, SB 213068, GI 262570, GW 1929, GW 7845, GW 0207, L-796449, indomethacin and ibuprofen.

A therapeutic composition prepared in accordance with a method according to the invention is thus suitable for inducing and / or amplifying the expression of MMR. Also, it can be considered as an immunostimulatory composition, capable of improving the level of alertness of the system. immune to many infectious microorganisms, to stimulate the innate immune response and to amplify the effector functions of macrophages when the latter are placed in the presence of these microorganisms. Also, advantageously and according to the invention, it is intended for the medication of immunocompromised subjects.

Advantageously, a therapeutic composition obtained by a manufacturing process in accordance with the invention, can also comprise one or more other active ingredients, in particular for providing a synergistic effect. In particular, in addition to a PPARγ agonist ligand, it can advantageously contain a quantity of wall extract of a microorganism against which it is sought to defend itself.

The dosage form of a therapeutic composition in accordance with the invention is chosen according to the route of administration and can be produced by traditional techniques. The invention also extends to a curative treatment of pathologies liable to be induced by microorganisms, of bacterial, fungal or protozoan origin, with mannosylated and / or fucosylated motifs. These include microorganisms such as: Streptococcus pneumoniae, Klebsellia pneumoniae, Cryptococcus neoformans, Mycobacterium tuberculosis, Candida albicans, Leishmania donovani, Pneumocystis carinii, Trypanosoma cruzi, Aspergillus fumigatus, Pseudomonion aeruginosas.

The invention is intended for persons likely to be exposed to these infectious agents, or who have been infected by them. It is intended, in particular and preferably, for patients suffering from immune deficiencies, innate or acquired, and who are therefore particularly sensitive to so-called opportunistic diseases; diseases which are mainly caused by fungal pathogens (for example, candidiasis), which are known to have a large number of mannosylated and / or fucosylated patterns on their surface.

In a treatment method according to the invention, the elimination of the pathogen essentially involves recognition of the pathogen mediated by the mannose receptors of macrophages whose expression has been induced and / or amplified by a ligand agonist of PPARγ, natural or synthetic. In one 2004/096195 - - -. ^" -

11 treatment method according to the invention, it advantageously implements a method of stimulating the expression of MMR as described above.

In particular, the patient is treated by administering an effective and suitable amount of at least one agonist ligand of PPARγ. This amount is, in general, at least equivalent to 1ΕC50 of the ligand (s) used (s).

Advantageously, a therapeutic composition is used, the manufacturing process of which has been presented above.

According to another implementation variant of the invention, the patient is transferred to the macrophages having been previously isolated from an intracorporeal medium, for example from a blood sample from this same patient or from a donor immunocompatible with this one ; these macrophages are then treated with PPARγ agonists in an extracorporeal culture medium, before being reinjected into said patient.

The invention also relates to a method of stimulating macrophages, a method of eliminating pathogens with mannosylated and / or fucosylated motifs, a method of manufacturing a therapeutic composition, characterized, in combination, by all or some of the characteristics below. above or below.

Other objects, characteristics and advantages of the invention will appear on reading the following experimental tests which refer to the appended figures, in which:

FIG. 1 is a graphic representation of the inhibitory effect of ligands agonists of PPARγ on the proliferation of C. albicans,

- Figure 2 is a graphic representation of the capacity of macrophages to phagocyte C. albicans depending on whether PPARγ is or is not inhibited, - Figure 3 shows fluorescence distribution spectra reflecting the induction of the mannose receptor by various agonist ligands of PPARγ ,

FIG. 4 shows fluorescence distribution spectra reflecting the involvement of PPARγ in regulating the expression of MMR, FIG. 5 is a graphic representation of the involvement of PPARγ in the regulation of the expression of MMR,

- Figure 6 is a graphic representation of the amplification effect of various PPARγ agonist ligands on the expression of MMR, - Figure 7 is a graphic representation of the in vivo effect of 15d-PGJ ₂ on the ability of macrophages to produce oxidizing agents,

FIG. 8 is a graphic representation of the level of expression of MMR in SWTSS and RAG-2 mice infected with C. albicans,

FIG. 9 is a graphic representation of the effect of the ligands agonists of PPARγ on the expression of MMR of homozygous (+ / +) and heterozygous (- / -) mice for the PPARγ gene,

- Figure 10 is a graphic representation of the in vivo effect of agonist ligands of PPARγ on the proliferation of C. albicans, in the gastrointestinal tract by C. albicans. • IN VITRO EXPERIMENTATIONS

The work carried out in vitro has made it possible to demonstrate a stimulation of the anti-infectious properties of macrophages vis-à-vis various microorganisms having on their surface mannosylated patterns - notably C. albicans-, via the increase in l phagocytic activity of macrophages and their ability to produce oxidizing agents.

It has also been shown that the receptor associated with the increase in phagocytosis of C. albicans by macrophages treated with specific ligands for PPARγ was indeed MMR. In addition, the use of a specific antisense for the mannose receptor has made it possible to show a direct implication of the induction of the expression of this receptor in the increase in the oxidative metabolism of macrophages. Finally, transfection experiments of a CMV-PPARγ reporter vector into RAW 264.7 cells, known to express weakly PPARγ, revealed a regulation of the expression of MMR by a particular signaling pathway in which the nuclear receptor PPARγ is involved. .

The materials and methods used are presented below. C. albicans strains were isolated from blood samples from a patient with candidiasis, using standard techniques.

Macrophages, for their part, come either from the peritoneum of SWISS female mice (Center d'Elevage, Etablissement Janvier, France), or from the peripheral blood of healthy donors. These macrophages, mouse or human, are cultivated in an SFM culture medium ("serum-free medium") optimized for the culture of macrophages, marketed by the company GibcoBRL, France. The adhesion of the cells occurs after approximately 2 hours of culture at 37 ° C. under an atmosphere at 5% CO ₂ . The cells which do not adhere to the culture dish are eliminated by rinsing with PBS (GibcoBRL, France). _

For the various tests, the adherent cells remaining on the dishes are stimulated by 15d-PGJ ₂ , ciglitazone or rosiglitazone (MERCK-LIPΗA, France) at different concentrations. To study the proliferation of C. albicans, measurements of incorporation of [HJuracile (AMERSHAM PHARMACIA BIOTECH, France) in RNA are carried out with cells in the process of multiplication.

For each test, 2.10 ⁵ macrophages per well are cultured for 2 hours on 96-well Falcon® plates, in SFM at 37 ° C. and under an atmosphere containing 5% CO ₂ . Macrophages are stimulated by PPARγ agonists for 24 hours, then infected with C. albicans blastoconidia in a macrophage / yeast proportion of 1: 200, for 6 hours at 37 ° C, under a humidified atmosphere containing 5% CO ₂ . For some tests, tritiated uracil (1 μCi) is added to each well. After a 6 hour challenge, the supernatants from the wells containing the non-adherent blastospores are collected and centrifuged. The pellet is washed with PBS. The radioactivity incorporated into the yeast RNA is counted with the BCS scintillation fluid (AMERSHAM PHARMACIA BIOTECH, France). The monolayers containing adherent or phagocytated yeasts are washed twice with PBS, then lysed with NaOH (IM) and neutralized with HCl (IM). The scintillation liquid is then added to each flask and the radioactivity is counted using a Pharmacia LKB1217 counter. The number of decays by minute (dpm) allows to assess the level of proliferation of C. albicans for each of the trials.

To study the mechanism of ingestion of C. albicans by macrophages, yeasts previously labeled with [HJuracile are used. Three colonies of C. albicans are dispersed in 1 ml of Sabouraud broth containing 150 μCi of [ ³ HJuracile. After 24 hours of culture at 37 ° C. with stirring, the labeled blastospores are centrifuged and resuspended in the SFM enriched with 1.8% glucose. For each test, the macrophage monolayers cultured on the 24-well plates are treated with PPARγ agonists for 20 hours, then infected with labeled and viable C. albicans (in a macropliage / yeast proportion of 1: 1) and left to incubate for 1 hour at 37 ° C, then 2 hours at 5 ° C. Submitted at 5 ° C, the phagocytosis mechanisms are blocked. At the end of each of the incubation periods (1 hour or 2 hours), the culture medium is removed, the macrophage monolayers are rinsed twice with PBS. The rinsing solutions are kept. The monolayers are lysed with NaOH (IM) and then neutralized with HCl (IM). The radioactivity of the supernatant and that of the rinsing solutions, as well as the radioactivity contained in the cell lysate are counted separately with the scintillation fluid BCS by means of a liquid scintillation counter. The phagocytosis index is evaluated as follows:

_n ,, Δ ( ₃₇ ^o _C _ ₅ ^o _C) dpm _single layers

% Phagocytosis ^:

Δ (35 ° C - 5 ° C) m _mθ nocouches + Φ ^m supernatant

For flow cytometry tests, the macrophages are rinsed with ice-cold PBS. The labeling of the MMRs is carried out for one hour, in ice, in the manner of a specific ligand conjugated with mannosylated bovine albumin marked with fluorescein FITC (840 nM) (SIGMA, France). In order to facilitate the selection of viable cell populations, staining with propidium iodide is carried out. All analyzes are performed by FACScalibur (Becton-Dickinson) using CellQuest software.

MMR cDNA analyzes by RT-PCR are carried out as follows. Total RNA is isolated from cells using reagent "Extract AU" (EUROBIO, France). The RNAs are reverse transcribed using the "First-Strand DNA Synthesis" kit (PROMEGA, France), during 30 amplification cycles (30 sec. At 95 ° C, 1 min at 48 ° C and 1 min at 68 ° VS). The primers sequences (sense MMR and antisense MMR) used to obtain the MMR cDNA are presented in Table 2.

Table 2

For the transfection tests, the macrophages of the murine cell line RAW 267.4 (QBiogene, France) are maintained in the exponential growth phase in DMEM supplemented with 10% PBS. The day before transfection, the cells are transferred to 6-well plates. The medium is then replaced by DMEM devoid of serum and the cells are transiently transfected for 18 hours at 60-80% confluence with the transfection reagent FuGENE 6 (ROCHE, Switzerland) containing the vector to be transfected (CMV-luciferase or CMV-PPARγ), in DNA / FuGENE 6 proportion of 1: 2, with a quantity of DNA of 3 μg. On the day of stimulation, the medium is replaced by the complete medium (DMEM + FBS 10%) containing the different molecules to be tested. The cells are collected 24 hours later and then analyzed by flow cytometry. The transfection of the CMV-luciferase vector (PROMEGA, France) serves as a control. The sequence pCMX-mPPARγ (Forman B.M. et al, Cell, 1995, 83: 803-812) codes for the murine PPARγ nuclear receptor.

Regarding the treatment with the ODN antisense, a sequence complementary to the 3 'untranslated region of the MMR mRNA (Yamamoto et al, Infect Immun, 1997, 65: 1077-82), the ODN antisense (100 nM) with 10 μg of cationic lipid Lipofectin (GBBCO, France) at 37 ° C for 5 min according to the Capacciolis et al. method. (Biochem Biophys Res Commun, 1998, 197: 818-25) then it is added to the macrophages cultivated in DMEM without serum. The cells are incubated for 4 hours at 37 ° C with 5% atmospheric CO ₂ . The medium is replaced by SFM containing the ODN antisense but without Lipofectin. The cultures are incubated for 3 days with a daily change of the medium. The concentration of the ODN antisense used does not cause any significant toxicity on the macrophages. Untreated macrophages and macrophages treated with Lipofectin alone serve as controls. The nucleic acid sequence of the ODN antisense is set out in table 2. The same treatment is carried out with a non-specific antisense of the MMR mRNA, the ANS antisense whose nucleic sequence is set out in table 2.

For the tests of production of oxidizing agents, the oxygen-dependent respiratory explosion of the macrophages treated by the PPARγ agonists is measured by chemiluminescence. The macrophages (2.10 ⁵ ) are transferred to culture dishes 5 mm in diameter. After activation of the cells, the production of oxidizing agents by the macrophages, in the presence or in the absence of C. albicans, is measured by chemiluminescence in the presence of luminol at 37 ° C. The chemiluminescence emission is recorded continuously for 1 hour after incubation of the cells with luminol (66 μM). To evaluate the production of the superoxide anion (O ₂ ^~ ), the chemiluminescence is measured in the presence of SOD, a specific inhibitor of the production of superoxide anion. To assess the production of nitric oxide (NO), chemiluminescence is measured in the presence of L-NMMA (NG-monomethyl-arginine, a specific competitor of L-arginine for the production of NO). None of the inhibitors affect viability at the concentrations used. Statistical analyzes are based on the measurement of the area under the curve and are expressed in strokes x seconds.

Test 1: Stimulation of the candistatic activity of murine peritoneal macrophages by the specific ligands of PPARγ.

The action of macrophages treated with specific ligands of PPARγ in the inhibition of proliferation of C. albicans is studied in the face of a 6 hour in vitro challenge with the strain 98-26135 (Coste A. et al, J. Antimicrobial Chemotherapy, 2002, 49: 731-740). The proliferation of C. albicans was determined in the presence of macrophages cultured using the radiometric technique. Figure 1 shows the inhibition of proliferation (in%) of C. albicans in the presence of macrophages not treated (C) or treated (T) with 15d-PGJ ₂ , ciglitazone (Cigli), and rosiglitazone (Rosi ).

Figure 1 shows that macrophages develop their own fungistatic activity, inhibiting the growth of C. albicans by 18%. In the presence of agonist ligands of PPARγ in the medium, the macrophages have a higher candistatic activity. In fact, the inhibition of proliferation is multiplied approximately by three (compared to the control) when the macrophages are treated with different drugs. PPARγ agonist ligands stimulate the candistatic activity of macrophages.

Trial 2: Increase in phagocytosis of C. albicans and in the production of oxidizing agents by ligands specific for PPARγ. As the defense mechanisms against C. albicans are essentially dependent on the capacity of macrophages to phagocyte yeasts and to exercise their fungicidal activity by producing toxic molecules (IROs and NO), the modulation of the phagocytosis of this yeast as well as the production oxidizing agents of macrophages treated with specific ligands of PPARγ, in the absence or presence of GW 9662 (CAYMAN CHEMICAL, Michigan, USA), an irreversible antagonist of PPARγ, were studied.

Figure 2 shows the phagocytosis capacity (expressed as a phagocytosis index) of peritoneal macrophages not treated (C) or treated with specific ligands of PPARγ (15d-PGJ ₂ , ciglitazone and rosiglitazone) in the absence or presence of GW 9662 , faced with a one hour challenge with C. albicans under the conditions of a macrophage / yeast ratio of 1: 1.

FIG. 2 shows that there is a significant increase in the number of phagocytic yeasts when the macrophages are treated with the agonist ligands of PPARγ. The number of yeasts internalized by macrophages is increased by 19% by treatment with 15d-PGJ ₂ , by 13% with ciglitazone (Cigli), 10% with rosiglitazone (Rosi), compared to untreated macrophages (C) which have a phagocytosis index of 46%.

In addition, the increase in the phagocytosis capacity of macrophages treated with PPARγ agonists is inhibited by GW 9662. The capacity of macrophages to produce superoxide and nitrogen monoxide and their modulation by 15d-PGJ ₂ have also been studied by the chemiluminescence method which makes it possible to evaluate not only the production of superoxide anions but also that of the peroxynitrite produced during the interaction between nitric oxide and the superoxide anion. In order to evaluate the production of superoxide anion, the chemiluminescence was measured - in the presence or not of SOD (enzyme metabolizing O ₂ ^~ into H ₂ 0 ₂ ) while the production of nitrogen monoxide is evaluated in the presence of an arginine competitor which is L-NMMA.

Table 3: Effect of PPARγ agonist ligands on the production of oxidizing agents by macrophages in the presence or absence of yeast. The evaluation of the production of oxidizing agents was determined on macrophages in culture by the chemiluminescence technique.

Table 3 shows that the macrophages, put in the presence of the different treatments, produce the same basal amount of oxidizing agents as the resident macrophages. The specific ligands of PPARγ alone (in the absence of C. albicans) do not induce an increase in the production of oxidizing agents by macrophages.

On the other hand, the addition of yeasts increases the production of oxidizing agents. PPARγ agonist ligands pre-activate the ability of macrophages to produce oxidizing agents. In response to C. albicans infection, macrophages in this activated state mainly produce superoxide anion. Indeed, in the presence of SOD, a specific inhibitor of production of superoxides, the production of oxidizing agents is significantly inhibited. Conversely, the chemiluminescence measured in the presence of L-NMMA is not inhibited. Thus, macrophages in this activation state do not produce nitric oxide. These results, which show that the PPARγ antagonist blocks the candistatic effects of the PPARγ ligands, suggest that PPARγ is involved in increasing the fungicidal activity of macrophages under the effect of the various treatments.

Test 3: Induction of the expression of mannose receptors by specific ligands of PPARγ.

As the mannose receptor is involved in phagocytosis C. albicans as well as many other pathogenic microorganisms with mannosylated residues on the outside of their walls, the expression of this receptor on the surface of macrophages treated with agonist ligands PPARγ was studied by flow cytometry thanks to the fixation of molecules of bovine mannosylated albumin (mBSA) labeled with fluorescein (FITC, fluorothioisocyanate).

The macrophages were cultured for 24 hours in the presence of 1 μM of 15d-PGJ ₂ , 5 μM of ciglitazone or 5 μM of rosiglitazone. The cells are then incubated in FITC-labeled mBSA and analyzed by flow cytometry. The distribution of the fluorescence of the cell population analyzed is shown in Figure 3.

FIG. 3 shows that the quantity of mannosylated bovine albumin labeled with fluorescein present on the macrophages is significantly increased when the cells are treated (T) with specific ligands of PPARγ in comparison with the untreated cells (C). The specificity of mBSA for the mannose receptor suggests that the specific ligands of PPARγ induce the expression of mannose receptors on the surface of macrophages. The regression analysis of the results set out in Figures 2 and

3 indicates a linear association between the quantity of MMR and the capacity of phagocytosis after treatment with the different PPARγ ligands of macrophages, thus demonstrating a true correlation between the expression of mannose receptors influenced by the different treatments and the capacity of these cells to internalize C. albicans (r = 0.942).

Test 4: Direct involvement of PPARγ in the regulation of the expression of mannose receptors by specific ligands of PPARγ.

In order to determine whether the transcription factor PPARγ is involved in the overexpression of the mannose receptor induced by the specific ligands for PPARγ, two different approaches were used. First, we transfected (for 18 hours) two reporter vectors, CMV-luciferase and CMV-PPARγ, where the luciferase gene and the PPARγ gene are dependent on a strong promoter, that of the cytomegalo virus. . We carried out these transfections in the RAW 264.7 line, known to weakly express PPARγ. After treatment with specific PPARγ ligands, the mannose receptors present on the cell surface were quantified by flow cytometry.

RAW 264.7 cells were then cultured for 24 hours in the presence of 1 μM of 15d-PGJ ₂ , 5 μM of ciglitazone or 5 μM of rosiglitazone. The cells are incubated with FITC-labeled mBSA and analyzed by flow cytometry. The distribution of the fluorescence of the cell population analyzed is shown in Figure 4 and the geometric mean of the corresponding fluorescence intensities, reported in Table 4 below.

Table 4 In a second step, the induction of the expression of the mannose receptor of murine peritoneal macrophages under the effect of the various treatments was studied by flow cytometry in the presence of an irreversible PPARγ antagonist, GW 9662. FIG. 4 shows that in RAW cells 264.7 control

(C), cells transfected with CMV-luciferase, treatment with PPARγ ligands has no effect on the expression of mannose receptors. Furthermore, the transfection of RAW 264.7 cells with the vector CMV-PPARγ restores the effect of the various treatments on the induction of the expression of mannose receptors. This demonstrates that PPARγ is directly involved in the regulation of the expression of the mannose receptor by specific ligands for PPARγ.

These results are confirmed by FIG. 5 which shows that the increase in the expression of the mannose receptor on the surface of the macrophages under the effect of 15d-PGJ ₂ is antagonized by GW 9662. To do this, the macrophages have were cultured 24 hours in the presence or absence of GW 9962 (0.1 μM), then incubated with mBSA labeled with FITC and analyzed by flow cytometry.

These two experiments show the involvement of PPARγ in the overexpression of mannose receptors on the surface of macrophages. Test 5: The induction of the expression of the mannose receptor on the surface of macrophages under the effect of different PPARγ agonists is dependent on transcriptional regulation.

In order to determine whether the activation of PPARγ leads to a transcriptional induction of the mannose receptor, two different techniques were carried out in parallel. To this end, a first approach consisted in transfecting a specific antisense of the mannose receptor mRNA into macrophages and the mannose receptors present on the surface of the cells were quantified by flow cytometry. At the same time, another technique was used and consisted in directly quantifying the expression of the mRNAs of the mannose receptor, under the effect of the various treatments, by quantitative RT-PCR in real time. To do this, the macrophages were treated for 3 days with the antisense specific for the mannose receptor mRNA (ODN). Macrophages are cultured for 24 hours in the presence of 1 μM of 15d-PGJ ₂ , 5 μM of ciglitazone, or 5 μM of rosiglitazone. The cells were then incubated with FITC-labeled mBSA, and then analyzed by flow cytometry. The intensity of the fluorescence analyzed is shown in Figure 6.

FIG. 6 shows that in the presence of the antisense, the amount of fluorescence is significantly inhibited whatever the treatments used. These results indicate on the one hand that the increase in the quantity of mBSA fixed on the macrophages results from an induction of the expression of the mannose receptors and, on the other hand, that this over-expression is the direct reflection of 'increased expression of mannose receptor mRNAs.

Trial 6: The mannose receptor is involved in the modulation of the oxidative functions of macrophages by the ligands of PPARγ. This study was carried out to determine whether the increase in the production of oxidizing agents under the effect of PPARγ agonists depended or not on the modulation of mannose receptors by these different treatments. For this purpose, the modulation of the production of oxidizing agents by macrophages transfected by the specific antisense of the mannose receptor mRNAs has been studied. After treatment with PPARγ agonists, the quantity of oxidizing agents produced by the transfected macrophages was assayed by chemiluminescence. The results are presented in Table 5.

Table 5: Involvement of the mannose receptor in the production of oxidizing agents for macrophages treated with PPARγ ligands. The production of oxidizing agents was determined on macrophages in culture by the chemiluminescence technique. C x S: strokes x seconds during lh. Macrophages are incubated for 3 days with a specific antisense (ODN) or with a non-specific antisense (ANS) of the mannose receptor mRNA.

Table 4 shows that the increase in the production of oxidizing agents by macrophages induced by the treatments is antagonized by the specific antisense of the mannose receptor mRNA With the non-specific antisense of the mannose receptor (ANS) no effect on the inhibition of the production of oxidizing agents is observed. Thus, the production of oxygenated free radicals is correlated with the induction of the expression of mannose receptors. Test 7: Effect of PPARγ ligands (rosiglitazone, ciglitazone, 15d-PGJ ₂ ) on the expression of mannose receptors in human monocytes.

The study was carried out on cultured human monocytes, prepared from peripheral blood mononuclear cells from healthy donors. The separation of mononuclear cells was carried out from pockets of cytopheresis residues, by a standard Ficoll-Hypaque gradient method (Bureau et al, J. Biol. Chem., 2001, 276: 23077-23083). The monocytes were isolated from the mononuclear cells, by adhesion after 2 hours of culture at 37 ° C. and under an atmosphere containing 5% of CO ₂ in a medium without serum for macrophages (SFM for macrophage from iNVITOGEN CORP., France) and supplemented with de_ la_ Lrglutamine .. Non-adherent cells were removed - by two washes with Hanks saline. The adherent cells (85% of the monocytes / macrophages) were cultured in SFM for macrophage.

It has been established, on these monocyte cultures, that the agonist ligands of PPARγ induce the expression of the mannose receptor after 18 hours of culture.

In addition, phagocytosis of C. albicans has been shown to be increased in proportion to the induction of mannose receptors.

Finally, it has been established that monocytes differentiated by PPARγ agonist ligands kill C. albicans by a mechanism dependent on the production of superoxide anions triggered during the interaction between C. albicans and mannose receptor.

• IN VIVO EXPERIMENTATIONS Following the work carried out obtained in vitro which showed that the treatment of macrophages with the ligands of PPARγ stimulated the antifungal activity via PPARγ, work was carried out in vivo in order to validate these results. The in vitro study was carried out on batches of 10 mice, either immunocompetent (SWISS) or immunodeficient (RAG-2), who were infected with C. albicans. These mice were treated, or not, with 15d-PGJ ₂ or with rosiglitazone.

Firstly, it was verified that the various treatments carried out in vivo did indeed modify the phagocytosis capacity of macrophages.

In a second step, the expression of the mannose receptor on the surface of macrophages extracted from mice, treated in vivo with 15d-PGJ ₂ or with rosiglitazone, was studied by flow cytometry. RAG-2 type immunodeficient mice (CNRS breeding center, Orléans, France) with a severe combined deficit in cell and humoral mediated immunity, do not have B and T lymphocytes, are used in typical viral pathology AIDS / HIV.

In this model using RAG-2, it was possible to reproduce orogastrointestinal candidiasis identical to that observed in immunocompromised patients. We thus examined, in vivo, the antifungal efficacy of this new therapeutic strategy. In parallel, this same protocol was carried out on immunocompetent SWISS mice (Breeding Center - Etablissement Janvier, France). The mice housed in sterile cages were infected by gavage of 500 μl of a yeast suspension (2.10 ⁷ cfu.mT ¹ ). This amount of yeast is sufficient to establish orogastrointestinal candidiasis in all mice.

Pharmacological doses of 15d-PGJ ₂ (0.01 μmol) or rosiglitazone (0.1 μmol) were injected intraperitoneally one day before infection with C. albicans. These injections were renewed every day for 5 days. 2004/096195

25

The macrophages were then collected by washing the peritoneal cavity of the infected mice treated or not, either with 15d-PGJ ₂ or with rosiglitazone. The macrophagic suspension was adjusted to 10 / ml in SFM (GibcoBRL, France) supplemented with glutamine, penicillin and streptomycin and was distributed at a rate of 1 ml per well in the microplates of 24 wells. After a 2 hour incubation in an oven at 37 ° C, 5% CO ₂ , the non-adherent cells were eliminated by two rinses in PBS. After these 2 hours, the cell mats in monolayers contain 95% of macrophages and are used to evaluate their phagocytosis capacity as well as their capacity to produce oxidizing agents.

In order to assess the capacity of macrophages to phagocyte C. albicans, a technique based on the incorporation of radioactive nucleotides (tritiated uracil) at the level of RNA of yeasts in the process of multiplication was carried out. The pre-labeled yeasts are placed on the macrophagic mats and incubated at 37 ° C under 5% C0 ₂ . After one hour of incubation, the radioactivity of the supernatants and of the rinses from the wells, as well as that contained in the cell lysates obtained by lysis with NaOH (IN) will be measured independently. Knowing that the radioactivity measured in the lysate is proportional to the quantity of phagocytated yeasts and that that measured in the supernatants ^" is proportional to the quantity of external yeasts, the percentage of phagocytated yeasts in relation to the quantity of total yeasts will be determined according to the formula next :

dpmiy _s at% Phagocytosis = dp ^m lys _a t + dpni _supernatant

In order to evaluate the production of oxidizing agents (oxygenated free radicals and nitrogen monoxide) by the macrophages after the cultivation of the macrophages, the joint activities of NADPH oxidase and of the inducible NOsynthase are measured by chemiluminescence in the presence of '' a chemiluminogenic probe, Luminol (SIGMA, France): 5-amino2,3-diydro-l, 4-phthalazine-dione. The production of chemiluminescence will be analyzed with a luminometer (Wallac Victor II, Finland) controlled by a computer. The luminol allows, in the presence of oxidizing agents, the emission of photons captured by the photomultiplier of the dark room of the luminometer thermostatically controlled at 37 ° C and transformed into quantifiable electrical pulses. The chemiluminescence values will be recorded continuously for 1 hour after incubation of the cells with luminol (66 μM) and in the presence or not of yeasts.

For MMR, the macrophages of mice infected with C. albicans and treated or not in vivo, either with 15d-PGJ ₂ or with rosiglitazone, were collected by washing the peritoneal cavity of these mice. The suspension is adjusted to 10 ⁶ / ml in SFM and incubated for 1 hour at 4 ° C. with mannosylated bovine albumin (mBSA) labeled with fluorescein (14 μM). The quantity of mBSA molecule fixed, direct reflection of the quantity of mannose receptors present on the cells, was analyzed using a flow cytometer (FACScalibur, Becton Dickinson).

Subsequently, it has been established that treatments of mice with PPARγ agonist ligands modify candidosal infection in several organs (esophagus, stomach, cecum, kidneys, liver). To do this, after infection with C. albicans, the SWISS and RAG-2 mice were treated with 15d-PGJ ₂ or with rosiglitazone, as previously described. During and at the end of the experiment, blood samples were taken in order to monitor the presence of yeasts in the blood of the mice. No samples showed the presence of yeasts in the blood, signifying that there was no spread of the fungal infection. At the end of the protocol, the mice were euthanized and the different organs were removed sterile in order to quantify the number of yeasts present in the different organs by CFU and quantitative PCR in real time.

To quantify the number of yeasts present in the various organs by CFU, after homogenization of each tissue, several dilutions are made and seeded in culture dishes. The dishes are incubated at 35 ° C for 24 to 48 hours and the number of colonies is then counted. To quantify the number of yeasts present in the different organs by PCR, after homogenization of the different organs, the total DNA is extracted by the phenol / chloroform method and subjected to a Q-PCR in time real. DNA quantification of C. albicans is carried out on a LightCycler machine (ROCHE MOLECULAR BlOCHEMICALS, Switzerland). The primers used were chosen from the conserved regions of the gene coding for the fungal 18S rRNA. The detection is done by hybridization of two labeled probes specific for C. albicans (fluorescence transfer).

Test 8: Stimulation of phagocytosis of C. albicans and of the production of oxidizing agents for macrophages by in vivo treatment with 15d-PGJ ₂ .

The study was carried out on immunocompetent or immunodeficient mice of the RAG-2 type (mice which have a severe combined deficit of cell and humoral mediated immunity, do not have B and T lymphocytes) treated or not, either by 15d-PGJ ₂ or by rosiglitazone, then infected with C. albicans (culture, by the radiometric technique, facing a one hour challenge with C. albicans, in a 1: 1 ratio).

Table 5 below shows the phagocytosis capacity of peritoneal macrophages taken from SWISS or RAG-2 mice infected with C. albicans, whether or not treated with 15d-PGJ ₂ in vivo. The results presented in this table show that the macrophages extracted from SWISS mice and RAG-2 mice, treated in vivo, either with 15d-PGJ ₂ or with rosiglitazone, exhibit a much greater phagocytosis capacity than the macrophages extracted from mice not treated in vivo (control). Thus, intraperitoneal administration of 15d-PGJ ₂ or rosiglitazone increases the ability of macrophages to internalize C. albicans.

Table 5

The capacity of macrophages to produce oxidizing agents was then determined, as well as the modulation of this production by in vivo treatment with 15d-PGJ ₂ or rosiglitazone. The evaluation of the production of oxidizing agents by macrophages, in the presence or in the absence of yeast, was determined on macrophages in culture by the chemiluminescence technique. Figure 7 shows the results obtained, in number of seconds (C x S) counted for 1 hour, for each test.

FIG. 7 shows that the macrophages extracted from SWISS and RAG-2 mice, treated in vivo with 15d-PGJ ₂ produce the same basal amount of oxidizing agents as the macrophages extracted from control mice. Treatment of mice with 15d-PGJ ₂ therefore does not induce an increase in the production of oxidizing agents by macrophages. However, the addition of yeasts increases the production of oxidizing agents.

Test 9: Amplification of the expression of mannose receptors by an in vivo treatment with 15d-PGJ ₂ or with rosiglitazone.

As the mannose receptor is involved in the phagocytosis of many pathogenic microorganisms such as in particular

C. albicans, the expression of this receptor on the surface of macrophages extracted from mice treated in vivo, with 15d-PGJ ₂ or with rosiglitazone, was studied by flow cytometry.

FIG. 8 shows that the quantity of mannosylated bovine albumin marked with fluorescein present on the macrophages is significantly increased when the cells are extracted from the mice treated in vivo with 15d-PGJ ₂ or with rosiglitazone. This suggests that the treatment of SWISS and RAG-2 mice, with 15d-PGJ ₂ or with rosiglitazone, induces an increase in the expression of mannose receptors on the surface of macrophages. Figure 9 shows that the induction of the mannose receptor by PPARγ ligands is greatly reduced in mice having a zero allele for the gene for this nuclear receptor (heterozygous mice, PPARγ ^{+ / "} ) compared to homozygous mice (PPARγ ^{+ / +} ). Thus, the effect of PPARγ ligands is more effective in mice which express the PPARγ gene on the two alleles of the chromosome, which suggests the role of PPARγ in the expression of the mannose receptor in vivo. The regression analysis of the results set out in FIG. 8 and in Table 5 indicates a linear association between the quantity of MMR on the surface of the macrophages and the phagocytosis capacity of these cells after treatment of the mice with 15d-PGJ ₂ or by rosiglitazone. This therefore demonstrates a real correlation between the expression of mannose receptors influenced by the treatments and the capacity of these cells to internalize C. albicans.

Test 10: Decrease in candidal infection by in vivo treatment with 15d-PGJ ₂ or with rosiglitazone.

In a second step, it was established that the treatments modified the candidosic infection at the level of several organs (esophagus, stomach, cecum, kidneys, liver). To do this, after infection with C. albicans, the SWISS and RAG-2 mice were treated either with 15d-PGJ ₂ or with rosiglitazone. During and at the end of the experiment, blood samples were taken in order to monitor the presence of yeasts in the blood of the mice. No samples showed the presence of yeasts in the blood, signifying that there was no spread of the fungal infection. At the end of the protocol, the mice were euthanized and the different organs were removed sterile in order to quantify the number of yeasts present in the different organs by CFU and quantitative PCR in real time. The results obtained on the esophagus (Œs.), The stomach (St.), and the cecum (Cas.), Are presented in Figure 10.

The two techniques for quantifying the number of yeasts show a significant reduction in the colonization of the various organs in mice treated, either with 15d-PGJ ₂ or with rosiglitazone, compared with untreated mice (C). In the liver and kidneys, no colonization by yeast is observed, thus showing an absence of dissemination of the fungal infection.

Claims

CLAIMS 1 / Method for manufacturing a therapeutic composition intended for the preventive or curative treatment of a pathology belonging to the group of pathologies induced by microorganisms, of bacterial, fungal or protozoan origin, which have, on the outer face of their walls, polysaccharides and / or lipopolysaccharides having at their free ends mannosylated and / or fucosylated units - with the exception of Mycobacterium avium-, characterized in that a therapeutically effective amount of at least one agonist ligand is used of the PPARγ nuclear receptor as an induction and / or amplification factor for the expression of mannose receptors of macrophages.

2 / A method of manufacturing a therapeutic composition according to claim 1, characterized in that said microorganisms are chosen from: Streptococcus pneumoniae, Klebsellia pneumoniae, Cryptococcus neoformans, Mycobacterium tuberculosis, Candida albicans, Leishmania donovani, Pneumocystis carinii, Trypanosoma cruzi , Aspergillus fumigatus, Pseudomonas aeruginosas, Legionella pneumophilla.

3 / A method of manufacturing a therapeutic composition according to one of claims 1 or 2, characterized in that at least one of the agonist ligands of PPARγ is chosen from the group of natural ligands: 15d-PGJ ₂ , prostaglandins A1 and D2, 9- and 13-hydoxyoctadecanoic acids, oxidized LDL derivatives.

4 / A method of manufacturing a therapeutic composition according to one of claims 1 or 2, characterized in that at least one of the agonist ligands of PPARγ is chosen from the group of synthetic ligands: troglitazone, rosiglitazone, pioglitazone , ciglitazone, englitazone, GW 347845, KRP-297, JTT-501β, SB 213068, GI 262570, GW 1929, GW 7845, GW 0207, L-796449, indomethacin and ibuprofen.

5 / A method of manufacturing a therapeutic composition according to one of claims 1 to 4, characterized in that said therapeutic composition is intended for the medication of immunocompromised subjects. 6 / A method of stimulating the expression of mannose receptors of macrophages in which, an effective amount of at least one agonist ligand of PPARγ is treated, a biological medium comprising macrophages.

II Stimulation method according to claim 6, characterized in that at least one of the agonist ligands of PPARγ is chosen from the group of natural ligands: 15d-PGJ ₂ , prostaglandins Al and D2, acids 9- and 13- hydoxyoctadecanoic, derivatives of oxidized LDL.

8 / A stimulation method according to claim 6, characterized in that at least one of the PPARγ agonist ligands is chosen from the group of synthetic ligands: troglitazone, rosiglitazone, pioglitazone, ciglitazone, englitazone, GW 347845, KRP-297, JTT-501β, SB 213068, GI 262570, GW 1929, GW 7845, GW 0207, L-796449, indomethacin and ibuprofen.

9 / A stimulation method according to one of claims 6 to 8, characterized in that said biological medium containing macrophages corresponds to an intracorporeal medium of a patient.

10 / Stimulation method according to one of claims 6 to 8, characterized in that one treats an extra-corporeal biological medium.

11 / A stimulation method according to claim 10, characterized in that the macrophages of said biological medium are prepared from a blood sample from a patient, then are treated with an effective amount of at least one agonist ligand of PPARγ, before being reinjected into the same patient.

12 / A stimulation method according to one of claims 9 or 11, characterized in that said patient suffers from immune deficiencies. 13 / stimulation method according to one of claims 9,

11 or 12, characterized in that said patient is subjected to a preventive or curative treatment of a pathology induced by microorganisms, of bacterial, fungal or protozoan origin, which have on the outer face of their walls polysaccharides and / or lipopolysaccharides having at their free ends mannosylated and / or fucosylated units. 14 / A stimulation method according to claim 13, characterized in that said microorganisms are chosen from: Streptococcus pneumoniae, Klebsellia pneumoniae, Cryptococcus neoformans, Mycobacterium tuberculosis, Candida albicans, Leishmania donovani, Pneumocystis carinii, Trypanosoma cruzi, Aspergillus fumatus aeruginosas, Legionella pneumophilla.

15 / Process for the elimination of microorganisms, of bacterial, fungal or protozoan origin, having, on the external face of their walls, polysaccharides and / or lipopolysaccharides with free mannosylated and / or fucosylated ends, characterized in that an effective amount of at least one PPARγ agonist ligand is placed in a medium infected with said microorganisms and in contact with macrophages as an induction and / or amplification factor for the expression of mannose receptors macrophages.

16 / A method of elimination according to claim 15, characterized in that said microorganisms are chosen from: Streptococcus pneumoniae, Klebsellia pneumoniae, Cryptococcus neoformans, Mycobacterium tuberculosis, Candida albicans, Leishmania donovani, Pneumocystis carinii, Trypanosoma cruzi, Aspergillus fumatus Pseudomonas aeruginosas, Legionella pneumophilla. 17 / elimination method according to one of claims 15 or 16, characterized in that at least one of the agonist ligands of PPARγ is chosen from the group of natural ligands: 15d-PGJ ₂ , prostaglandins Al and D2, 9- and 13-hydoxyoctadecanoic acids, oxidized LDL derivatives.

18 / elimination method according to one of claims 15 or 16, characterized in that at least one of the agonist ligands of PPARγ is chosen from the group of synthetic ligands: troglitazone, rosiglitazone, pioglitazone, ciglitazone, englitazone, GW 347845, KRP-297, JTT-501β, SB 213068, GI 262570, GW 1929, GW 7845, GW 0207, L-796449, indomethacin and ibuprofen . 19 / Method for the curative treatment of a pathology induced by microorganisms, of bacterial, fungal or protozoan origin, which have, on the outer face of their walls, polysaccharides and / or lipopolysaccharides with mannosylated and / or fucosylated motifs at their free ends, in which the elimination of said microorganism is favored by recognition of the antigen mediated by the mannose receptors macrophages whose expression has been induced and / or has been amplified by a PPARγ agonist ligand.

20 / A method of curative treatment according to claim 19, characterized in that said microorganisms are chosen from: Streptococcus pneumoniae, Klebsellia pneumoniae, Ciyptococcus neoformans, Mycobacterium tuberculosis, Candida albicans, Leishmania donovani, Pneumocystis carinii, Trypanosoma cruzi, Aspergillus Pseudomonas aeruginosas, Legionella pneumophilla.

21 / Method of curative treatment according to one of claims 19 or 20, characterized in that a stimulation method according to one of claims 6 to 14 is implemented.

22 / Curative treatment method according to one of claims 19 or 20, characterized in that an elimination method according to one of claims 15 to 18 is implemented.

23 / A method of curative treatment according to one of claims 19 to 22, characterized in that a therapeutic composition made in accordance with a method according to one of claims l to 5 is used.